Chromatographic analysis on optical bio-discs and methods relating thereto

ABSTRACT

Preparation and analysis of biomedical samples is performed using an optical bio-disc system. More specifically, chromatography, including for example, affinity, size exclusion, reverse phase, and ion exchange may be performed using an optical bio-disc system. Ion exchange chromatography may include anion exchange, cation exchange, cation exchange linked immunoassays (CELIA), and anion exchange linked immunoassays, in conjunction with calorimetric and/or fluorescent detection and quantization using an optical analysis disc or optical bio-disc. Improved methods for preparing assays, methods for depositing the reagents required for the assays, discs for performing assays, and detection systems are described herein.

REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.60/449,192, filed Feb. 21, 2003, which is hereby incorporated byreference in its entirety, including FIGS. 1-22C.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates in general to biochemical assays. Morespecifically, but without restriction to the particular embodimentshereinafter described in accordance with the best mode of practice, thepresent invention relates to methods and apparatus for chromatographybased immunochemical assays performed on optical bio-discs and relateddetection systems.

2. Description of the Related Art

The detection and quantification of analytes in the blood or other bodyfluids are essential for diagnosis of diseases, elucidation of thepathogenesis, and for monitoring the response to drug treatment.Traditionally, diagnostic assays are performed in laboratories bytrained technicians using complex apparatus. Performing these assays isusually time-consuming and costly. Thus, there is a significant need tomake diagnostic assays and forensic assays of all types faster and morelocal to the end-user. Ideally, clinicians, patients, investigators, themilitary, other health care personnel, and consumers should be able totest themselves for the presence of certain risk factors or diseaseindicators in their systems, and to test for the presence of certainbiological material at a crime scene or on a battlefield. At present,there are a number of medical diagnostic, silicon-based, devices withnucleic acids and/or proteins attached thereto that are commerciallyavailable or under development. These chips are not for use by theend-user, or for use by persons or entities lacking very specializedexpertise and expensive equipment.

Commonly assigned U.S. Pat. No. 6,030,581 entitled “Laboratory in aDisk” issued Feb. 29, 2000 (the '581 patent) is hereby incorporated byreference in its entirety. The '581 patent discloses an apparatus thatincludes an optical disc, adapted to be read by an optical reader, whichhas a sector having a substantially self-contained assay system usefulfor localizing and detecting an analyte suspected of being in a sample.U.S. Pat. No. 5,993,665, issued Nov. 30, 1999 (the '665 patent) entitled“Quantitative Cell Analysis Methods Employing Magnetic Separation”discloses analysis of biological specimens in a fluid medium where thespecimens are rendered magnetically responsive by immuno-specificbinding with ferromagnetic colloid. The '665 patent is herebyincorporated by reference in its entirety.

SUMMARY OF THE INVENTION

The present invention relates to performing chromatography, includingfor example, affinity, size exclusion, reverse phase, and ion exchange.Ion exchange chromatography may include anion exchange, cation exchange,cation exchange linked immunoassays (CELIA), and anion exchange linkedimmunoassays, in conjunction with colorimetric and/or fluorescentdetection and quantitation using an optical analysis disc or opticalbio-disc. The invention includes methods for preparing assays, methodsfor depositing the reagents required for the assays, discs forperforming assays, and detection systems.

High pressure liquid chromatography (HPLC) and other types ofchromatography is generally used to separate substances or analytes ofinterest having different physical properties and quantitate theseanalytes using UV/VIS, IR, luminescence, or fluorescence detection.Chromatographic instruments generally require costly equipment andmaintenance and trained personnel to carry out complicatedtime-consuming tests. It is an object of the present invention to makepossible a simple chromatography system for testing analytes, portableand for use by the end user.

The present invention includes methods for isolating and quantifying theconcentration of an analyte of interest in a biological sample onoptical bio-discs using calorimetric or fluorometric detection. Analytesmay include, for example, Hemoglobin, glycated and non-glycatedhemoglobin, and other isoforms of proteins. All reagents necessary forthe assays may be immobilized on the optical disc prior to the assay. Toperform the assay, the sample (preferably serum, but other types of bodyfluids could also be used) is loaded into the channel via the injectionport. After injection, the port is sealed, the disc is spun, and thesample is moved through one or more micro-chromatographic matrices, bycentrifugation, comprising different separation media including, forexample, size exclusion and ion exchange matrices. The matrix may beformed from resins or beads, gels, or membranes. Once the analyte ofinterest is separated chromatographically, the analyte solution,containing the analyte of interest is then directed into an analysischamber. The analysis chamber may contain detection reagents including,but not limited to, capture agents bound to the surface of a capturezone and signal antibodies conjugated with one or more reporters, bothof which have affinity to different epitopes on the same analyte ofinterest. Reporters may include, but are not limited to, fluorophores,luminophores, microspheres, enzymes, and nanospheres. The analyte isincubated in the analysis chamber at a pre-determined temperature andtime to allow sufficient binding of the analyte to the capture agent andbinding of the signal antibodies to the analyte. After incubation theanalysis chamber is washed to remove unbound signal antibodies andanalytes. If the reporter used in the assay is a non-enzyme detectablereporter such as beads, then the analysis chamber may then be analysedfor presence and amount of reporter beads using the disc reader.Otherwise, if an enzyme reporter is used, an enzyme substrate is addedto the analysis chamber. The enzyme is allowed to catalize anenzyme-substrate reaction that produces a detectable signal such ascolor or fluorescence. The optical disc reader then quantifies theintensity of the color of fluorescence developed. After approximately 3minutes of data collection and processing, the results of the assay aredisplayed on a computer monitor. Alternatively, an inherent enzymaticactivity of the analyte itself may be advantageously used to produce adetectable signal. A non-limiting example of such an analyte ishemoglobin that has an inherent peroxidase activity. Thus capture andsignal agents are not necessary with this method thereby allowing a onestep assay method without the need for washing steps. In this method,the sample is loaded into the disc, ran through the matrix, and into theanalysis chamber, as described above. The analysis chamber, in thismethod, would only contain the appropriate substrate, a peroxidasesubstrate like ABTS (2,2′-azino-di-[3-ethyl-benzthiazoline] sulfonicacid) may be used in conjunction with the hemoglobin analyte, forexample. Once the signal is generated, the disc is then analyzed usingthe optical disc reader, as described above.

It should be noted that most diagnostic calorimetric assays in clinicallaboratories are carried out at 37 degrees Celsius to facilitate andaccelerate color development. For ease of operation, colorimetric assaysperformed on optical discs are optimized to run at ambient temperature.The optimization includes selection of enzyme sources, enzymesconcentrations, and sample preparation.

Chromagen selection, in the calorimetric aspect of the presentinvention, is of critical importance in optimizing colorimetric assaysfor optical density measurements on bio-discs since chromagens need tobe detected at specific wavelengths. CD-R type disc readers, forexample, require chromagens that can be detected in the infrared region(750 nm to 800 nm). Other types of optical disc systems may be used inthe present invention including DVD, DVD-R, fluorescent, phosphorescent,and any other similar optical disc reader. The amplitude of opticaldensity measurements depends on the optical path length, the molarextinction coefficient of the chromagen and the concentration of theanalyte of interest (Beer's law). To optimize the sensitivity ofcalorimetric assays on optical discs, several chromagens with high molarextinction coefficients at the wavelengths of interest have beenidentified and evaluated.

Chromagens suitable for colorimetric assays on CD-R type optical discsinclude, but are not limited to, N,N′-Bis(2-hydroxy-3-sulfopropyl)tolidine, disodium salt (SAT-3),N-(Carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)

-   -   -diphenylamine sodium salt (DA-64),        2,2′-azino-dimethylthiozoline-6-sulfonate (ABTS), Trinder's        reagents N-Ethyl-N-(2-hydroxy-3-sulfopropyl)₃-methylaniline,        sodium salt, dihydrate (TOOS) with the coupling reagent        3-(N-Methyl-N-phenylamino)-6-aminobenzenesulfonic acid, and        sodium salt (NCP-11).

According to one aspect of the present invention, there are provideddetection methods for quantifying the concentration of an analyte ofinterest in a biological sample on the bio-discs. The detection includesdirecting a beam of electromagnetic energy from a disc drive toward thecapture field and analyzing electromagnetic energy returned from ortransmitted through the capture field.

The optical density change, in the colorimetric assay aspect of thepresent invention, may be quantified by the optical disc reader by tworelated ways. These include measuring the change in light eitherreflected or transmitted. The disc may be referred to as reflective,transmissive, or some combination of reflective and transmissive. In areflective disc, an incident light beam is focused onto the disc(typically at a reflective surface where information is encoded),reflected, and returned through optical elements to a detector on thesame side of the disc as the light source. In a transmissive disc, lightpasses through the disc (or portions thereof) to a detector on the otherside of the disc from the light source. In a transmissive portion of adisc, some light may also be reflected and detected as reflected light.Different detection systems are used for different types of bio-discs(top versus bottom detector).

The conversion of data captured by the CD reader into meaningfulconcentration units is mediated via data processing software specificfor the assay of interest.

The apparatus and methods in embodiments of the present invention can bedesigned for use by an end-user, inexpensively, without specializedexpertise and expensive equipment. The system can be made portable, andthus usable in remote locations where traditional diagnostic equipmentmay not generally be available.

Alternatively, fluorescent assays can be carried out to quantify theconcentration of an analyte of interest in a biological sample on theoptical discs. In this case, the energy source in the disc drivepreferably has a wavelength controllable light source and a detectorthat is or can be made specific to a particular wavelength. In yetanother alternative, a disc drive can be made with a specific lightsource and detector to produce a dedicated device, in which case thesource may only need fine-tuning.

Analysis of biological fluids aimed at the quantitative and qualitativedetermination of substances associated with a wide variety ofphysiological disorders, bioresearch, proteomics, environmental studies,agriculture, and food industry, relies on specific binding assays fromwhich the immunoassay plays a dominant role. The outstanding specificityand sensitivity for quantitative determination of an almost limitlessnumber of analytes in practically any milieu, and the ability tominiaturize and adapt to automation makes them ideal tools for routineassays.

Antibody binding techniques are based on the interaction of a bindingantibody, receptor, or other binding proteins with an antigen or aspecific ligand molecule and the formation of an antibody-antigen orreceptor-ligand complex. By changing certain conditions a binding assaycan be designed to determine either an analyte, ligand, or targetbinding reagent or an antibody of interest. The steps are similar butthe assay configuration provides results pertinent to the antigen orantibody of interest.

1. Capture Probe Binding and Sample Application

When a sample is injected into a micro-channel, fluidic circuit, or flowchannel on an optical bio-disc, the target agent including, for example,target antigen or antibody, binds to a capture probe bound in a captureor target zone on a solid support such as a disc substrate. The captureprobe may be an antigen recognized by the target antibody or an antibodyor receptor with specific affinity to the target antigen or ligand.Following the binding step, unbound target agent is removed through awash step. It should be understood that various techniques, proceduresand chemistries, know in the art, may be used to bind the capture probeonto a solid support including, but not limited to, direct covalentbinding of probes onto a metallic or activated surface, passiveadsorption, and through cross-linking reagents.

Further details relating to surface chemistries used to bind probes ontosolid support are disclosed in, for example, the above incorporatedcommonly assigned co-pending U.S. Provisional Application Ser. No.60/353,770 entitled “Capture Layer Assemblies Including Metal Layer forImmobilization of Receptor Molecules and Related Optical Assay Discs”filed Jan. 30, 2002; and U.S. Provisional Application Ser. No.60/353,745 entitled “Capture Layer Assemblies Including PolymerSubstrates for Immobilization of Receptor Molecules and Related OpticalAssay Discs” filed Jan. 30, 2002.

In addition to surface chemistries for attaching capture probes,blocking agents may be used to block areas within the capture or targetzone and the flow channel where capture probes are not bound(non-capture areas) to prevent non-specific binding of the target oranalyte, signal probes, and reporters onto these areas. Blocking agentsinclude, but are not limited to proteins such as BSA, gelatin, sugarssuch as sucrose, detergents such as tween-20, genetic material such assheared salmon sperm DNA, and polyvinyl alcohol.

2. Signal Generation

Signal is generated from tags or labels attached to signal or reporteragents or probes that have specific affinity to a target agent. Signalagents or probes may include, for example, signal antibodies or signalligands, tagged with microspheres, sub-micron nanospheres, or enzymes.The microspheres or nanospheres may be fluorescent labeled(fluospheres), phosphorescent, luminecent, or chemiluminescent. Themicrospheres or nanospheres may also carry different chemicalfunctionalities including, for example, carboxyl, amino, aldehyde, andhydrazine functional groups. These functional groups may facilitatebinding of the signal agent. The enzyme may facilitate a chemicalreaction that produces fluorescence, color, or a detectable signal inthe presence of a suitable substrate. For example, conjugatedhorseradish peroxidase (HRP; Pierce, Rockford, Ill.) may be used withthe substrate 3,3,5,5-tetramethylbenzidine (TMB; Calbiochem cat. no.613548, CAS-54827-17-7) in the presence of hydrogen peroxide to producean insoluble precipitate. Horseradish peroxidase can also be used inconjunction with CN/DAB (4-chloronaphthol/3,3′-diaminobenzidine,tetrahydrochloride), 4-CN (4-chloro-1-napthol), AEC (3-amino-9-ethylcarbazol) and DAB (3,3-diaminobenzidine tetrahydrochloride) to forminsoluble precipitates. Similarly, the enzyme alkaline phosphatase (AP)can be used with the substrate bromochloroindolylphosphate in thepractice of the present invention. Other suitable enzyme/substratecombinations will be apparent to those of skill in the art.

3. Detection

The signal from the microspheres or the enzyme reaction can be read withthe optical bio-disc readers developed to be utilized in conjunctionherewith. Either a bottom detector on a disc with a reflective cover, ora top detector with a transmissive disc may be employed as the opticalbio-disc reader for the assay and disc inventions disclosed herein.

(a) Disc Implementation

The assays and methods of the present invention may be advantageouslyimplemented on an analysis disc, modified optical disc, or bio-disc. Thebio-disc may include a flow channel having target or capture zone, areturn channel in fluid communication therewith, a mixing chamber influid communication with the flow channel, and in some embodiments awaste reservoir in fluid communication with the flow channel.

The bio-disc may be implemented on an optical disc including aninformation encoding format such as CD, CD-R, or DVD or a modifiedversion thereof. The bio-disc may include encoded information forperforming, controlling, and post-processing the test or assay. Forexample, such encoded information may be directed to controlling therotation rate of the disc, incubation time, incubation temperature,and/or specific steps of the assay. Depending on the test, assay, orinvestigational protocol, the rotation rate may be variable withintervening or consecutive sessions of acceleration, constant speed, anddeceleration. These sessions may be closely controlled both as to speedand time of rotation to provide, for example, mixing, agitation, orseparation of fluids and suspensions with agents, reagents, DNA, RNA,antigen, antibodies, ligands, and receptors.

(b) Drive Implementation

A bio-disc drive assembly or reader may be employed to rotate the disc,read and process any encoded information stored on the disc, and analyzethe samples in the flow channel of the bio-disc. The bio-disc drive isthus provided with a motor for rotating the bio-disc, a controller forcontrolling the rate of rotation of the disc, a processor for processingreturn signals from the disc, and an analyzer for analyzing theprocessed signals. The drive may include software specifically developedfor performing the assays disclosed herein.

The rotation rate of the motor is controlled to achieve the desiredrotation of the disc. The bio-disc drive assembly may also be utilizedto write information to the bio-disc either before or after the testmaterial in the flow channel and target or capture zone is interrogatedby the read beam of the drive and analyzed by the analyzer. The bio-discmay include encoded information for controlling the rotation rate of thedisc, providing processing information specific to the type of test tobe conducted, and for displaying the results on a display monitorassociated with the bio-drive in accordance with the assay methodsrelating hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects of the present invention together with additionalfeatures contributing thereto and advantages accruing therefrom will beapparent from the following description of the preferred embodiments ofthe invention which are shown in the accompanying drawing figures withlike reference numerals indicating like components throughout, wherein:

FIG. 1 is a pictorial representation of a bio-disc system;

FIG. 2 is an exploded perspective view of a reflective bio-disc;

FIG. 3 is a top plan view of the disc shown in FIG. 2;

FIG. 4 is a perspective view of the disc illustrated in FIG. 2 withcut-away sections showing the different layers of the disc;

FIG. 5 is an exploded perspective view of a transmissive bio-disc;

FIG. 6 is a perspective view representing the disc shown in FIG. 5 witha cut-away section illustrating the functional aspects of asemi-reflective layer of the disc;

FIG. 7 is a graphical representation showing the relationship betweenthickness and transmission of a thin gold film;

FIG. 8 is a top plan view of the disc shown in FIG. 5;

FIG. 9 is a perspective view of the disc illustrated in FIG. 5 withcut-away sections showing the different layers of the disc including thetype of semi-reflective layer shown in FIG. 6;

FIG. 10 is a perspective and block diagram representation illustratingthe system of FIG. 1 in more detail;

FIG. 11 is a partial cross sectional view taken perpendicular to aradius of the reflective optical bio-disc illustrated in FIGS. 2, 3, and4 showing a flow channel formed therein;

FIG. 12 is a partial cross sectional view taken perpendicular to aradius of the transmissive optical bio-disc illustrated in FIGS. 5, 8,and 9 showing a flow channel formed therein and a top detector;

FIG. 13 is a partial longitudinal cross sectional view of the reflectiveoptical bio-disc shown in FIGS. 2, 3, and 4 illustrating a wobble grooveformed therein;

FIG. 14 is a partial longitudinal cross sectional view of thetransmissive optical bio-disc illustrated in FIGS. 5, 8, and 9 showing awobble groove formed therein and a top detector;

FIG. 15 is a view similar to FIG. 11 showing the entire thickness of thereflective disc and the initial refractive property thereof;

FIG. 16 is a view similar to FIG. 12 showing the entire thickness of thetransmissive disc and the initial refractive property thereof;

FIG. 17A is an exploded perspective view of a reflective bio-discincorporating equi-radial channels of the present invention;

FIG. 17B is a top plan view of the disc shown in FIG. 17A;

FIG. 17C is a perspective view of the disc illustrated in FIG. 17A withcut-away sections showing the different layers of the equi-radialreflective disc;

FIG. 18A is an exploded perspective view of a transmissive bio-discutilizing the e-radial channels of the present invention;

FIG. 18B is a top plan view of the disc shown in FIG. 18A;

FIG. 18C is a perspective view of the disc illustrated in FIG. 18A withcut-away sections showing the different layers of this embodiment of theequi-radial transmissive bio-disc;

FIG. 19 is a pictorial representation of images derived from atransmissive optical bio-disc showing differences in signal derived fromvarious concentrations of the hemoglobin;

FIG. 20 is a graphical representation of a dose response curve generatedusing the optical bio-disc system of the present invention;

FIG. 21 is a top plan view of another embodiment of the optical bio-dischaving a micro-chromatographic matrix in a fluidic circuit;

FIG. 22A are top plan views of various layers of a chromatographicoptical bio-disc of the present invention;

FIG. 22B is an exploded perspective view of the chromatographic opticalbio-disc of FIG. 22A; and

FIG. 22C is a partial cross sectional view taken perpendicular to aradius of the optical bio-disc illustrated in FIG. 22B showing thedirection sample flow within the fluidic circuit.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates in general to preparation of biomedicalsamples and analysis of same using an optical bio-disc system. Morespecifically, this invention is directed to performing chromatography,including for example, affinity, size exclusion, reverse phase, and ionexchange. Ion exchange chromatography may include anion exchange, cationexchange, cation exchange linked immunoassays (CELIA), and anionexchange linked immunoassays, in conjunction with calorimetric and/orfluorescent detection and quantitation using an optical analysis disc oroptical bio-disc. The invention includes methods for preparing assays,methods for depositing the reagents required for the assays, discs forperforming assays, and detection systems. Each of the aspects of thepresent invention is discussed below in further detail.

Drive System and Related Discs

FIG. 1 is a perspective view of an optical bio-disc 110 according to thepresent invention as implemented to conduct the cell counts anddifferential cell counts disclosed herein. The present optical bio-disc110 is shown in conjunction with an optical disc drive 112 and a displaymonitor 114. Further details relating to this type of disc drive anddisc analysis system are disclosed in commonly assigned and co-pendingU.S. patent application Ser. No. 10/008,156 entitled “Disc Drive Systemand Methods for Use with Bio-discs” filed Nov. 9, 2001 and U.S. patentapplication Ser. No. 10/043,688 entitled “Optical Disc Analysis SystemIncluding Related Methods For Biological and Medical Imaging” filed Jan.10, 2002, both of which are herein incorporated by reference.

FIG. 2 is an exploded perspective view of the principal structuralelements of one embodiment of the optical bio-disc 110. FIG. 2 is anexample of a reflective zone optical bio-disc 110 (hereinafter“reflective disc”) that may be used in the present invention. Theprincipal structural elements include a cap portion 116, an adhesivemember or channel layer 118, and a substrate 120. The cap portion 116includes one or more inlet ports 122 and one or more vent ports 124. Thecap portion 116 may be formed from polycarbonate and is preferablycoated with a reflective surface 146 (FIG. 4) on the bottom thereof asviewed from the perspective of FIG. 2. In the preferred embodiment,trigger marks or markings 126 are included on the surface of thereflective layer 142 (FIG. 4). Trigger markings 126 may include a clearwindow in all three layers of the bio-disc, an opaque area, or areflective or semi-reflective area encoded with information that sendsdata to a processor 166, as shown FIG. 10, that in turn interacts withthe operative functions of the interrogation or incident beam 152, FIGS.6 and 10.

The second element shown in FIG. 2 is an adhesive member or channellayer 118 having fluidic circuits 128 or U-channels formed therein. Thefluidic circuits 128 are formed by stamping or cutting the membrane toremove plastic film and form the shapes as indicated. Each of thefluidic circuits 128 includes a flow channel 130 and a return channel132. Some of the fluidic circuits 128 illustrated in FIG. 2 include amixing chamber 134. Two different types of mixing chambers 134 areillustrated. The first is a symmetric mixing chamber 136 that issymmetrically formed relative to the flow channel 130. The second is anoff-set mixing chamber 138. The off-set mixing chamber 138 is formed toone side of the flow channel 130 as indicated.

The third element illustrated in FIG. 2 is a substrate 120 includingtarget or capture zones 140. The substrate 120 is preferably made ofpolycarbonate and has a reflective layer 142 deposited on the topthereof, FIG. 4. The target zones 140 are formed by removing thereflective layer 142 in the indicated shape or alternatively in anydesired shape. Alternatively, the target zone 140 may be formed by amasking technique that includes masking the target zone 140 area beforeapplying the reflective layer 142. The reflective layer 142 may beformed from a metal such as aluminum or gold.

FIG. 3 is a top plan view of the optical bio-disc 110 illustrated inFIG. 2 with the reflective layer 142 on the cap portion 116 shown astransparent to reveal the fluidic circuits 128, the target zones 140,and trigger markings 126 situated within the disc.

FIG. 4 is an enlarged perspective view of the reflective zone typeoptical bio-disc 110 according to one embodiment of the presentinvention. This view includes a portion of the various layers thereof,cut away to illustrate a partial sectional view of each principal layer,substrate, coating, or membrane. FIG. 4 shows the substrate 120 that iscoated with the reflective layer 142. An active layer 144 is appliedover the reflective layer 142. In the preferred embodiment, the activelayer 144 may be formed from polystyrene. Alternatively, polycarbonate,gold, activated glass, modified glass, or modified polystyrene, forexample, polystyrene-co-maleic anhydride, may be used. In addition,hydrogels can be used. Alternatively as illustrated in this embodiment,the plastic adhesive member 118 is applied over the active layer 144.The exposed section of the plastic adhesive member 118 illustrates thecut out or stamped U-shaped form that creates the fluidic circuits 128.The final principal structural layer in this reflective zone embodimentof the present bio-disc is the cap portion 116. The cap portion 116includes the reflective surface 146 on the bottom thereof. Thereflective surface 146 may be made from a metal such as aluminum orgold.

Referring now to FIG. 5, there is shown an exploded perspective view ofthe principal structural elements of a transmissive type of opticalbio-disc 110 according to the present invention. The principalstructural elements of the transmissive type of optical bio-disc 110similarly include the cap portion 116, the adhesive or channel member118, and the substrate 120 layer. The cap portion 116 includes one ormore inlet ports 122 and one or more vent ports 124. The cap portion 116may be formed from a polycarbonate layer. Optional trigger markings 126may be included on the surface of a thin semi-reflective layer 143, asbest illustrated in FIGS. 6 and 9. Trigger markings 126 may include aclear window in all three layers of the bio-disc, an opaque area, or areflective or semi-reflective area encoded with information that sendsdata to the processor 166, FIG. 10, which in turn interacts with theoperative functions of the interrogation beam 152, FIGS. 6 and 10.

The second element shown in FIG. 5 is the adhesive member or channellayer 118 having fluidic circuits 128 or U-channels formed therein. Thefluidic circuits 128 are formed by stamping or cutting the membrane toremove plastic film and form the shapes as indicated. Each of thefluidic circuits 128 includes the flow channel 130 and the returnchannel 132. Some of the fluidic circuits 128 illustrated in FIG. 5include the mixing chamber 134. Two different types of mixing chambers134 are illustrated. The first is the symmetric mixing chamber 136 thatis symmetrically formed relative to the flow channel 130. The second isthe off-set mixing chamber 138. The off-set mixing chamber 138 is formedto one side of the flow channel 130 as indicated.

The third element illustrated in FIG. 5 is the substrate 120, which mayinclude the target or capture zones 140. The substrate 120 is preferablymade of polycarbonate and has the thin semi-reflective layer 143deposited on the top thereof, FIG. 6. The semi-reflective layer 143associated with the substrate 120 of the disc 110 illustrated in FIGS. 5and 6 is significantly thinner than the reflective layer 142 on thesubstrate 120 of the reflective disc 110 illustrated in FIGS. 2, 3 and4. The thinner semi-reflective layer 143 allows for some transmission ofthe interrogation beam 152 through the structural layers of thetransmissive disc as shown in FIGS. 6 and 12. The thin semi-reflectivelayer 143 may be formed from a metal such as aluminum or gold.

FIG. 6 is an enlarged perspective view of the substrate 120 andsemi-reflective layer 143 of the transmissive embodiment of the opticalbio-disc 110 illustrated in FIG. 5. The thin semi-reflective layer 143may be made from a metal such as aluminum or gold. In the preferredembodiment, the thin semi-reflective layer 143 of the transmissive discillustrated in FIGS. 5 and 6 is approximately 100-300 Å thick and doesnot exceed 400 Å. This thinner semi-reflective layer 143 allows aportion of the incident or interrogation beam 152 to penetrate and passthrough the semi-reflective layer 143 to be detected by a top detector158, FIGS. 10 and 12, while some of the light is reflected or returnedback along the incident path. As indicated below, Table 1 presents thereflective and transmissive characteristics of a gold film relative tothe thickness of the film. The gold film layer is fully reflective at athickness greater than 800 Å. While the threshold density fortransmission of light through the gold film is approximately 400 Å.

In addition to Table 1, FIG. 7 provides a graphical representation ofthe inverse relationship of the reflective and transmissive nature ofthe thin semi-reflective layer 143 based upon the thickness of the gold.Reflective and transmissive values used in the graph illustrated in FIG.7 are absolute values. TABLE 1 Au Film Reflection and Transmission(Absolute Values) Thickness (Angstroms) Thickness (nm) ReflectanceTransmittance 0 0 0.0505 0.9495 50 5 0.1683 0.7709 100 10 0.3981 0.5169150 15 0.5873 0.3264 200 20 0.7142 0.2057 250 25 0.7959 0.1314 300 300.8488 0.0851 350 35 0.8836 0.0557 400 40 0.9067 0.0368 450 45 0.92220.0244 500 50 0.9328 0.0163 550 55 0.9399 0.0109 600 60 0.9448 0.0073650 65 0.9482 0.0049 700 70 0.9505 0.0033 750 75 0.9520 0.0022 800 800.9531 0.0015

With reference next to FIG. 8, there is shown a top plan view of thetransmissive type optical bio-disc 110 illustrated in FIGS. 5 and 6 withthe transparent cap portion 116 revealing the fluidic channels, thetrigger markings 126, and the target zones 140 as situated within thedisc.

FIG. 9 is an enlarged perspective view of the optical bio-disc 110according to the transmissive disc embodiment of the present invention.The disc 110 is illustrated with a portion of the various layers thereofcut away to show a partial sectional view of each principal layer,substrate, coating, or membrane. FIG. 9 illustrates a transmissive discformat with the clear cap portion 116, the thin semi-reflective layer143 on the substrate 120, and trigger markings 126. In this embodiment,trigger markings 126 include opaque material placed on the top portionof the cap. Alternatively the trigger marking 126 may be formed byclear, non-reflective windows etched on the thin reflective layer 143 ofthe disc, or any mark that absorbs or does not reflect the signal comingfrom the trigger detector 160, FIG. 10. FIG. 9 also shows the targetzones 140 formed by marking the designated area in the indicated shapeor alternatively in any desired shape. Markings to indicate target zone140 may be made on the thin semi-reflective layer 143 on the substrate120 or on the bottom portion of the substrate 120 (under the disc).Alternatively, the target zones 140 may be formed by a masking techniquethat includes masking the entire thin semi-reflective layer 143 exceptthe target zones 140. In this embodiment, target zones 140 may becreated by silk screening ink onto the thin semi-reflective layer 143.In the transmissive disc format illustrated in FIGS. 5, 8, and 9, thetarget zones 140 may alternatively be defined by address informationencoded on the disc. In this embodiment, target zones 140 do not includea physically discernable edge boundary.

With continuing reference to FIG. 9, an active layer 144 is illustratedas applied over the thin semi-reflective layer 143. In the preferredembodiment, the active layer 144 is a 10 to 200 μm thick layer of 2%polystyrene. Alternatively, polycarbonate, gold, activated glass,modified glass, or modified polystyrene, for example,polystyrene-co-maleic anhydride, may be used. In addition, hydrogels canbe used. As illustrated in this embodiment, the plastic adhesive member118 is applied over the active layer 144. The exposed section of theplastic adhesive member 118 illustrates the cut out or stamped U-shapedform that creates the fluidic circuits 128.

The final principal structural layer in this transmissive embodiment ofthe present bio-disc 110 is the clear, non-reflective cap portion 116that includes inlet ports 122 and vent ports 124.

Referring now to FIG. 10, there is a representation in perspective andblock diagram illustrating optical components 148, a light source 150that produces the incident or interrogation beam 152, a return beam 154,and a transmitted beam 156. In the case of the reflective bio-discillustrated in FIG. 4, the return beam 154 is reflected from thereflective surface 146 of the cap portion 116 of the optical bio-disc110. In this reflective embodiment of the present optical bio-disc 110,the return beam 154 is detected and analyzed for the presence of signalelements by a bottom detector 157. In the transmissive bio-disc format,on the other hand, the transmitted beam 156 is detected, by a topdetector 158, and is also analyzed for the presence of signal elements.In the transmissive embodiment, a photo detector may be used as a topdetector 158.

FIG. 10 also shows a hardware trigger mechanism that includes thetrigger markings 126 on the disc and a trigger detector 160. Thehardware triggering mechanism is used in both reflective bio-discs (FIG.4) and transmissive bio-discs (FIG. 9). The triggering mechanism allowsthe processor 166 to collect data only when the interrogation beam 152is on a respective target zone 140. Furthermore, in the transmissivebio-disc system, a software trigger may also be used. The softwaretrigger uses the bottom detector to signal the processor 166 to collectdata as soon as the interrogation beam 152 hits the edge of a respectivetarget zone 140. FIG. 10 further illustrates a drive motor 162 and acontroller 164 for controlling the rotation of the optical bio-disc 110.FIG. 10 also shows the processor 166 and analyzer 168 implemented in thealternative for processing the return beam 154 and transmitted beam 156associated the transmissive optical bio-disc.

As shown in FIG. 11, there is presented a partial cross sectional viewof the reflective disc embodiment of the optical bio-disc 110 accordingto the present invention. FIG. 11 illustrates the substrate 120 and thereflective layer 142. As indicated above, the reflective layer 142 maybe made from a material such as aluminum, gold or other suitablereflective material. In this embodiment, the top surface of thesubstrate 120 is smooth. FIG. 11 also shows the active layer 144 appliedover the reflective layer 142. As also shown in FIG. 11, the target zone140 is formed by removing an area or portion of the reflective layer 142at a desired location or, alternatively, by masking the desired areaprior to applying the reflective layer 142. As further illustrated inFIG. 11, the plastic adhesive member 118 is applied over the activelayer 144. FIG. 11 also shows the cap portion 116 and the reflectivesurface 146 associated therewith. Thus when the cap portion 116 isapplied to the plastic adhesive member 118 including the desired cutoutshapes, flow channel 130 is thereby formed. As indicated by thearrowheads shown in FIG. 11, the path of the incident beam 152 isinitially directed toward the substrate 120 from below the disc 110. Theincident beam then focuses at a point proximate the reflective layer142. Since this focusing takes place in the target zone 140 where aportion of the reflective layer 142 is absent, the incident continuesalong a path through the active layer 144 and into the flow channel 130.The incident beam 152 then continues upwardly traversing through theflow channel to eventually fall incident onto the reflective surface146. At this point, the incident beam 152 is returned or reflected backalong the incident path and thereby forms the return beam 154.

FIG. 12 is a partial cross sectional view of the transmissive embodimentof the bio-disc 110 according to the present invention. FIG. 12illustrates a transmissive disc format with the clear cap portion 116and the thin semi-reflective layer 143 on the substrate 120. FIG. 12also shows the active layer 144 applied over the thin semi-reflectivelayer 143. In the preferred embodiment, the transmissive disc has thethin semi-reflective layer 143 made from a metal such as aluminum orgold approximately 100-300 Angstroms thick and does not exceed 400Angstroms. This thin semi-reflective layer 143 allows a portion of theincident or interrogation beam 152, from the light source 150, FIG. 10,to penetrate and pass upwardly through the disc to be detected by a topdetector 158, while some of the light is reflected back along the samepath as the incident beam but in the opposite direction. In thisarrangement, the return or reflected beam 154 is reflected from thesemi-reflective layer 143. Thus in this manner, the return beam 154 doesnot enter into the flow channel 130. The reflected light or return beam154 may be used for tracking the incident beam 152 on pre-recordedinformation tracks formed in or on the semi-reflective layer 143 asdescribed in more detail in conjunction with FIGS. 13 and 14. In thedisc embodiment illustrated in FIG. 12, a physically defined target zone140 may or may not be present. Target zone 140 may be created by directmarkings made on the thin semi-reflective layer 143 on the substrate120. These marking may be formed using silk screening or any equivalentmethod. In the alternative embodiment where no physical indicia areemployed to define a target zone (such as, for example, when encodedsoftware addressing is utilized) the flow channel 130 in effect may beemployed as a confined target area in which inspection of aninvestigational feature is conducted.

FIG. 13 is a cross sectional view taken across the tracks of thereflective disc embodiment of the bio-disc 110 according to the presentinvention. This view is taken longitudinally along a radius and flowchannel of the disc. FIG. 13 includes the substrate 120 and thereflective layer 142. In this embodiment, the substrate 120 includes aseries of grooves 170. The grooves 170 are in the form of a spiralextending from near the center of the disc toward the outer edge. Thegrooves 170 are implemented so that the interrogation beam 152 may trackalong the spiral grooves 170 on the disc. This type of groove 170 isknown as a “wobble groove”. A bottom portion having undulating or wavysidewalls forms the groove 170, while a raised or elevated portionseparates adjacent grooves 170 in the spiral. The reflective layer 142applied over the grooves 170 in this embodiment is, as illustrated,conformal in nature. FIG. 13 also shows the active layer 144 appliedover the reflective layer 142. As shown in FIG. 13, the target zone 140is formed by removing an area or portion of the reflective layer 142 ata desired location or, alternatively, by masking the desired area priorto applying the reflective layer 142. As further illustrated in FIG. 13,the plastic adhesive member 118 is applied over the active layer 144.FIG. 13 also shows the cap portion 116 and the reflective surface 146associated therewith. Thus, when the cap portion 116 is applied to theplastic adhesive member 118 including the desired cutout shapes, theflow channel 130 is thereby formed.

FIG. 14 is a cross sectional view taken across the tracks of thetransmissive disc embodiment of the bio-disc 110 according to thepresent invention as described in FIG. 12, for example. This view istaken longitudinally along a radius and flow channel of the disc. FIG.14 illustrates the substrate 120 and the thin semi-reflective layer 143.This thin semi-reflective layer 143 allows the incident or interrogationbeam 152, from the light source 150, to penetrate and pass through thedisc to be detected by the top detector 158, while some of the light isreflected back in the form of the return beam 154. The thickness of thethin semi-reflective layer 143 is determined by the minimum amount ofreflected light required by the disc reader to maintain its trackingability. The substrate 120 in this embodiment, like that discussed inFIG. 13, includes the series of grooves 170. The grooves 170 in thisembodiment are also preferably in the form of a spiral extending fromnear the center of the disc toward the outer edge. The grooves 170 areimplemented so that the interrogation beam 152 may track along thespiral. FIG. 14 also shows the active layer 144 applied over the thinsemi-reflective layer 143. As further illustrated in FIG. 14, theplastic adhesive member or channel layer 118 is applied over the activelayer 144. FIG. 14 also shows the cap portion 116 without a reflectivesurface 146. Thus, when the cap is applied to the plastic adhesivemember 118 including the desired cutout shapes, the flow channel 130 isthereby formed and a part of the incident beam 152 is allowed to passtherethrough substantially unreflected.

FIG. 15 is a view similar to FIG. 11 showing the entire thickness of thereflective disc and the initial refractive property thereof. FIG. 16 isa view similar to FIG. 12 showing the entire thickness of thetransmissive disc and the initial refractive property thereof. Grooves170 are not seen in FIGS. 15 and 16 since the sections are cut along thegrooves 170. FIGS. 15 and 16 show the presence of the narrow flowchannel 130 that is situated perpendicular to the grooves 170 in theseembodiments. FIGS. 13, 14, 15, and 16 show the entire thickness of therespective reflective and transmissive discs. In these figures, theincident beam 152 is illustrated initially interacting with thesubstrate 120 which has refractive properties that change the path ofthe incident beam as illustrated to provide focusing of the beam 152 onthe reflective layer 142 or the thin semi-reflective layer 143.

Alternative embodiments of the bio-disc according to the presentinvention will now be described with reference to FIGS. 17A, 17B, 17C,18A, 18B, and 18C. Various features of the discs of these latterembodiments have been already illustrated with reference to FIGS. 1 to16, and therefore such common features will not be described again inthe following. Accordingly, and for the sake of simplicity, as a generalrule in FIGS. 17 and 18, only the features differentiating the bio-disc110 from those of FIGS. 1 to 21 are represented.

Furthermore, the following description of the bio-disc of the inventioncan be readily applied to a transmissive-type as well as to areflective-type optical bio-disc described above in conjunction withFIGS. 2 to 9.

FIG. 17A is an exploded perspective view of a reflective bio-discincorporating equi-radial channels 200 of the present invention. Thisgeneral construction corresponds to the radial-channel disc shown inFIG. 2. The e-rad or eRad implementation of the bio-disc 110 shown inFIG. 17A similarly includes the cap 116, the channel layer 118, and thesubstrate 120. The channel layer 118 includes the equi-radial fluidchannels 200, while the substrate 120 includes the corresponding arraysof target zones 140.

FIG. 17B is a top plan view of the disc shown in FIG. 17A. FIG. 17Bfurther shows a top plan view of an embodiment of eRad disc with atransparent cap portion, which disc has two tiers of circumferentialfluid channels with ABO chemistry and two blood types (A+ and AB+). Asshown in FIG. 17B, it is also possible to provide a priori, at themanufacturing stage of the disc of the invention, a plurality of entryports, eventually at different radial coordinate, so that a range ofequi-radial, spiralling, or radial reaction sites and/or channels arepossible on one disc. These channels can be used for different testsuites, or for multiple samples of single test suites.

FIG. 17C is a perspective view of the disc illustrated in FIG. 17A withcut-away sections showing the different layers of the e-radialreflective disc. This view is similar to the reflective disc shown inFIG. 4. The e-rad implementation of the reflective bio-disc shown inFIG. 17C similarly includes the reflective layer 142, active layer 144as applied over the reflective layer 142, and the reflective layer 146on the cap portion 116.

FIG. 18A is an exploded perspective view of a transmissive bio-discutilizing the e-radial channels of the present invention. This generalconstruction corresponds to the radial-channel disc shown in FIG. 5. Thetransmissive e-rad implementation of the bio-disc 110 shown in FIG. 18Asimilarly includes the cap 116, the channel layer 118, and the substrate120. The channel layer 118 includes the equi-radial fluid channels 200,while the substrate 120 includes the corresponding arrays of targetzones 140.

FIG. 18B is a top plan view of the transmissive e-rad disc shown in FIG.18A. FIG. 18B further shows two tiers of circumferential fluid channelswith ABO chemistry and two blood types (A+ and AB+). As previouslydiscussed, the assays are performed in the target, capture, or analysiszones 140.

FIG. 18C is a perspective view of the disc illustrated in FIG. 18A withcut-away sections showing the different layers of this embodiment of thee-rad transmissive bio-disc. This view is similar to the transmissivedisc shown in FIG. 9. The e-rad implementation of the transmissivebio-disc shown in FIG. 18C similarly includes the thin semi-reflectivelayer 143 and the active layer 144 as applied over the thinsemi-reflective layer 143.

Detection of Hemoglobin and Glycohemoglobin Using the Optical Bio-Disc

Glycohemoglobin analysis is used in long-term carbohydrate control ofdiabetics. Glycohemoglobin is formed when glucose binds to hemoglobin(Hb) at the N-terminal valine on the beta-chain resulting in theformation of HbAlc. Antibody-based assays have been used to detect thenon-enzymatic glycation of Hb directly. However, producing HbAlcspecific antibodies in animals is very difficult since the sugar moietyof the glycohemoglobin molecule is not exposed and will rarely result ina specific immuneresponse. A combination of isocratic ion exchangechromatography with a class-specific immunoassay for hemoglobin canrapidly analyze glycated hemoglobin without the need of a specific probefor HbAlc. Different methods for glycohemoglobin analysis implemented onthe optical bio-discs are described below.

Cation Exchange Linked Immunoassay (CELIA) on the Optical Bio-Disc IonExchange Resins

A sandwich immunoassay for hemoglobin was developed by immobilizinghaptoglobin (a general capture agent for hemoglobin species) directly onthe gold surface of an optical bio-disc substrate. Horseradishperoxidase (HRP)-labeled goat anti-human hemoglobin antibody was used asthe enzyme conjugated signal antibody. ABTS(2,2′-azino-di-(3-ethyl-benzthiazoline sulfonic acid) was used as theenzyme substrate. Optical bio-disc derived images were taken andfour-parameter-fitted standard curves were generated as shown in FIGS.19 and 20. The results indicate that the optical bio-disc assay issensitive for hemoglobin and is capable of detecting both glycated andnon-glycated hemoglobin species to the same degree.

Weak cation exchange resins (e.g., carboxymethyl Sephadex beads) may beused to separate non-glycated hemoglobin from glycated hemoglobinspecies in a test sample. FIG. 21 illustrates an embodiment of theoptical bio-disc of the present invention wherein weak cation exchangebeads 203 are integrated into the fluidic circuit 128 to form amicro-chromatographic matrix 204 in the optical bio-disc 110 to isolatedesired analytes including glycated hemoglobin, for example. In thismethod, a hemoglobin sample (e.g. blood lysate), containing bothglycated and non-glycated forms of hemoglobin, is loaded into the inletport 122. The disc 110 is then spun thereby moving the sample throughthe cation exchange micro-chromatographic matrix 204. The non-glycatedhemoglobin binds to the beads 203 and only the glycated hemoglobinleaves the matrix 204 and moves through a filter 214 and into ananalysis or assay zone 202 where the analyte is quantified as describedabove. Alternatively, the non-glycated hemoglobin may be isolated usinganionic beads. In this alternative embodiment, glycated hemoglobin bindto the anionic beads while the non-glycated hemoglobin passes throughthe micro-chromatographic matrix 204 and is quantified. The totalhemoglobin also needs to be quantified along with either the glycated ornon-glycated hemoglobin to determine the percentage of glycatedhemoglobin. The total hemoglobin may be quantified directly using thesample loaded directly into the analysis zone 202 or neutral beads mayalso be used in the micro-chromatographic matrix 204 wherein both formsof hemoglobin can freely pass through thereby allowing quantitation ofthe total hemoglobin.

Fluorescent labels may be used instead of HRP-labeled anti humanhemoglobin signal antibodies and the assay quantified using afluorescent optical bio-disc drive. Furthermore the capture and signalagents may be haptoglobin instead of antibodies. In this case, the assaywill consist of a haptoglobin capture agent immobilized on a capture ortarget zone within an analysis chamber and a HRP- or fluorescent labeledhaptoglobin signal agent. Other detectable labels known in the art canalso be applied. The pseudo-peroxidase activity of hemoglobin can alsobe used to produce a detectable signal with the appropriate peroxidasesubstrate and requires only the (unlabeled) haptoglobin capture agent(or other capture proteins for hemoglobin) to capture the analyte, asdescribed above.

The ion exchange matrix may be packed into the fluidic channels andseparated from the analysis chamber 202 by using a different channeland/or chamber thickness for the analysis chamber 202. For example,40-120 micron cation exchange beads may be used to form the ion exchangematrix. Thus a channel or chamber on the disc with a thickness of >120microns (“ion exchange zone”) connected to a second channel or chamberwith a thickness of <40 microns (analysis chamber) can be used. Thenarrower thickness of the analysis chamber prevents the beads fromentering the analysis chamber. Furthermore a microfluidic channel designwith a capillary valve system can also be used in conjunction with theion exchange linked immunoassay embodiments of the present invention.

Ion Exchange Membranes

1) Lateral Flow Membranes

FIGS. 22A, 22B, and 22C show an optical bio-disc 110 for use in themembrane chromatographic assay of the present invention whereinchemically modified membranes 216 having binders directed to eitherglycated or non-glycated hemoglobin, for example, may be used as thematrix material of the present invention. In this case the lateral flowmembrane 216 may be formed, for example, from carboxymethyl (a weakcation) membranes, for binding non-glycated hemoglobin.

In a sandwich assay format method of the present invention, the captureagent, which can be an antibody or haptoglobin or another captureprotein for hemoglobin, may be labeled with reporter particles (latexbeads, gold beads, carbon beads, or others). After sample applicationand disc spinning steps, non-glycated hemoglobin binds to the cationexchange matrix and glycated hemoglobin will move to the specificanalysis chamber and to the target or capture zone. The target zone isthen analyzed for the presence and amount of reporter particles usingthe optical bio-disc reader. For the measurement of non-glycatedhemoglobin the ion exchange matrix may be formed from a weak anionexchange membrane.

2) Flow Through Membrane (Membrane Adsorbers)

Ion Exchange Membrane Adsorbers used in ready-to-use filters (Sartorius,Goettengen, Germany) may also be used to form the matrix. Furthermore,centrifuge based Ion Exchange Membrane Spin Columns such as for exampleVivapure (Viva Science, Hannover, Germany) can also be embedded into anoptical bio-disc and used for the separation of different isoforms ofproteins (including various hemoglobin species) with subsequent,immunoassay-based optical bio-disc detection.

With reference to FIG. 22A, there is shown different layers of thebio-disc 110 for use in the lateral flow and flow through membrane basedassays of the present invention. In this embodiment, several layers maybe assembled to form the spiral fluidic circuit 128 as shown. Theselayers may include a top cover disc or cap portion 116 (illustrated inFIG. 22B), an upper channel layer 208, a lower channel layer 212, amiddle membrane or chromatography layer 210 situated between upper layer208 and lower layer 212, and a bottom substrate layer 120. Substratelayer 120 may be the transmissive or reflective type substrate 120 asdiscussed above. The top cap portion 116 includes one or more inletports 122 and one or more vent ports 124 as shown in FIGS. 2, 5, 17A,and 22B. The chromatography layer 210 includes pass through ports 206formed therein. The chemically modified membranes 216 are may be placedover the pass through ports 206. The upper 208 and lower 212 channellayers have fluidic circuits 128 formed therein such that when the disc110 is assembled with the chromatography layer 210 placed between theupper 208 and lower 212 channel layers, and the bottom substrate layer120 and top cap portion 116 are accordingly bonded to the disc; a spiralfluidic chromatographic circuit is formed.

Referring now to FIG. 22B, there is depicted an exploded view of thebio-disc 110 described above in conjunction with FIG. 22A showing thevarious layers of the bio-disc including the top cap portion 116, theupper channel layer 208, the chromatography layer 210, the lower channellayer 212, and the bottom substrate layer 120.

Turning next to FIG. 22C, there is illustrated a partial cross sectionof a fully assembled bio-disc as described in FIG. 22A showing thedirection of fluid flow (arrows) through the fluidic circuit 128. Sampleis introduced into the disc 110 through the inlet port 122 of the capportion 116. The upper channel layer 208, chromatography layer 210, andlower channel layer 212 are positioned such that fluid is directedthrough a series of chemically modified membranes 216 as the fluid orsample moves through the fluidic circuit 128 as illustrated. Thechemically modified membrane 216 may include for example the IonExchange and Lateral Flow Membranes described above.

Bioseparation with a porous membrane is of critical importance inmolecular biology assays. The present application demonstrates fluidicchannel arrangements for integration of porous materials, such as aporous membrane or a chromatographic membrane, into the optical bio-disc110.

The bio-disc 110 is preferably made from several layers of polycarbonatediscs and patterned adhesives to form a fluidic circuit. By integratingthe porous membrane, the applied analyte will flow through the porousmaterial when the analyte is driven by centrifugal and/or other types offorces.

With continuing reference to FIG. 22C, there is shown a pattern for eachlayer of a disc for use in biochemical assays. The optical bio-disc 110of the present invention may include the following layers:

1. Substrate Layer 120 is a lens disc with signal tracks. The substratelayer may be a CD, CD-R, DVD, or DVD-R type disc, for example. Thesubstrate 120 may include a reflective layer 142 which can betransmissive or partially reflective as described above in conjunctionwith FIGS. 2-9. Thus, it can be used to track disc spinning and provideenough optical signal for detection.

2. Lower channel layer 212 may be formed from an adhesive with fluidicchannels 128 formed therein.

3. Chromatographic layer 210 is a disc layer having pass through ports206 designed such that a chromatographic membrane material 216 may beintegrated into the optical bio-disc 110. Chromatographic membranes 216are preferably placed in or on the pass through ports 206. The membraneand chromatographic layer thickness are preferably identical. If thethickness of the membrane and chromatographic layer is different, thenthickness of each can be adjusted by applying multiple layers.

4. Upper channel layer 208 may be formed from an adhesive with fluidicchannels formed therein. The patterned fluidic channels overlap with thefluidic channels from the lower channel layer 212 at the pass throughports 206 of the chromatographic layer only, as shown. Thus, the analytewill pass through these fluidic paths by vertically flowing through themembranes only, as shown in FIG. 22C

5. The topmost cap portion 116 is a cover disc. The fluidic channels 128are made to accommodate the test sample, especially when a large analytevolume is required for the assay.

6. The optical bio-disc of the present invention may optionally includea sealing layer (not shown) over the cap portion 116. It covers the ventport 124 and inlet port 122 and prevents contamination of the fluidiccircuits 128 and also prevents evaporation of the test sample whenloaded into the bio-disc.

Generally, the separation concept is based on having the chromatographicmembrane material 216 arranged within the two layers of fluidic path asshown in FIG. 22C. Furthermore, bioseparation can be achieved byproperly arranging the fluidic path to allow the analyte to flow througha series of chromatographic membranes 216.

FIG. 22C shows one segment of the integration arrangement and thisdesign module can be scaled-up or scaled-down, by considering suchfactors as: membrane size, numbers of membrane required, and requireddisc space.

By extending this module in series, the analyte can flow through morethan two layers of membrane (as shown in FIG. 22). The present inventionmay be used for hemoglobin separation using a cation exchange membrane.The present invention may also be used in various bioseparationapplications which are different from separation by porous sizing only.

More particular discussion of membranes as implemented on opticalbio-discs are provided in the following commonly assigned and co-pendingprovisional applications: U.S. Provisional App. Ser. No. 60/353,740entitled “Methods and Apparatus for Separation of Lipoproteins UsingMembranes on Optical Bio-Discs” filed Jan. 30, 2002; U.S. ProvisionalApp. Ser. No. 60/353,300 entitled “Methods for Differential Cell CountsIncluding Leukocytes and Use of Optical Bio-Disc for Performing Same”filed Jan. 31, 2002; U.S. Provisional App. Ser. No. 60/353,948 entitled“Methods for Quantitation and Multiplexing of Receptor Ligand Assay byUse of Ultrathin Biomembranes Including Modified Optical Disc and Drive”filed Jan. 31, 2002; U.S. Provisional App. Ser. No. 60/354,014 entitled“Membrane Assays Implemented On Optical Analysis Disc” filed Jan. 31,2002; U.S. Provisional App. Ser. No. 60/353,913 entitled “OpticalBio-Disc Membrane Quantification Apparatus and Methods Using ControlLines as Internal Standard” filed Jan. 31, 2002; U.S. Provisional App.Ser. No. 60/353,818 entitled “Methods And Apparatus For Separation OfBlood Using Membranes On Optical Bio-Discs” filed Jan. 31, 2002; U.S.Provisional App. Ser. No. 60/354,319 entitled “Use of Avidin-BiotinSystems for Increase Detection Sensitivity in Membrane Based Assays andRelated Optical Analysis Disc” filed Feb. 4, 2002; and U.S. ProvisionalApp. Ser. No. 60/354,379 entitled “Application Methods For Bio-MembraneAssays In Bio-Disc System and Optical Analysis Disc Made AccordingThereto” filed Feb. 4, 2002. All of these applications are hereinincorporated by reference in their entireties. They thus providebackground and related disclosure as support hereof as if fully repeatedherein.

Additional embodiments, aspects, details, and attributes of the presentinvention are disclosed in Appendix A and B, appended hereto. Theseappendices are therefore a part hereof wherein more specificallyAppendix A includes pages A1-A38, and Appendix B includes pages B1-B23.

3) Other Implementations of the Current Invention

This invention or different aspects thereof may be readily implementedin, adapted to, or employed in combination with the discs, assays, andsystems disclosed in the following commonly assigned and co-pendingpatent applications: U.S. patent application Ser. No. 09/378,878entitled “Methods and Apparatus for Analyzing Operational andNon-operational Data Acquired from Optical Discs” filed Aug. 23, 1999;U.S. Provisional Patent Application Ser. No. 60/150,288 entitled“Methods and Apparatus for Optical Disc Data Acquisition Using PhysicalSynchronization Markers” filed Aug. 23, 1999; U.S. patent applicationSer. No. 09/421,870 entitled “Trackable Optical Discs with ConcurrentlyReadable Analyte Material” filed Oct. 26, 1999; U.S. patent applicationSer. No. 09/643,106 entitled “Methods and Apparatus for Optical DiscData Acquisition Using Physical Synchronization Markers” filed Aug. 21,2000; U.S. patent application Ser. No. 09/999,274 entitled “OpticalBiodiscs with Reflective Layers” filed Nov. 15, 2001; U.S. patentapplication Ser. No. 09/988,728 entitled “Methods and Apparatus forDetecting and Quantifying Lymphocytes with Optical Biodiscs” filed Nov.16, 2001; U.S. patent application Ser. No. 09/988,850 entitled “Methodsand Apparatus for Blood Typing with Optical Bio-discs” filed Nov. 19,2001; U.S. patent application Ser. No. 09/989,684 entitled “Apparatusand Methods for Separating Agglutinants and Disperse Particles” filedNov. 20, 2001; U.S. patent application Ser. No. 09/997,741 entitled“Dual Bead Assays Including Optical Biodiscs and Methods RelatingThereto” filed Nov. 27, 2001; U.S. patent application Ser. No.09/997,895 entitled “Apparatus and Methods for Separating Components ofParticulate Suspension” filed Nov. 30, 2001; U.S. patent applicationSer. No. 10/005,313 entitled “Optical Discs for Measuring Analytes”filed Dec. 7, 2001; U.S. patent application Ser. No. 10/006,371 entitled“Methods for Detecting Analytes Using Optical Discs and Optical DiscReaders” filed Dec. 10, 2001; U.S. patent application Ser. No.10/006,620 entitled “Multiple Data Layer Optical Discs for DetectingAnalytes” filed Dec. 10, 2001; U.S. patent application Ser. No.10/006,619 entitled “Optical Disc Assemblies for Performing Assays”filed Dec. 10, 2001; U.S. patent application Ser. No. 10/020,140entitled “Detection System For Disk-Based Laboratory and ImprovedOptical Bio-Disc Including Same” filed Dec. 14, 2001; U.S. patentapplication Ser. No. 10/035,836 entitled “Surface Assembly forImmobilizing DNA Capture Probes and Bead-Based Assay Including OpticalBio-Discs and Methods Relating Thereto” filed Dec. 21, 2001; U.S. patentapplication Ser. No. 10/038,297 entitled “Dual Bead Assays IncludingCovalent Linkages for Improved Specificity and Related Optical AnalysisDiscs” filed Jan. 4, 2002; U.S. patent application Ser. No. 10/043,688entitled “Optical Disc Analysis System Including Related Methods forBiological and Medical Imaging” filed Jan. 10, 2002; U.S. ProvisionalApplication Ser. No. 60/348,767 entitled “Optical Disc Analysis SystemIncluding Related Signal Processing Methods and Software” filed Jan. 14,2002 U.S. patent application Ser. No. 10/086,941 entitled “Methods forDNA Conjugation Onto Solid Phase Including Related Optical Biodiscs andDisc Drive Systems” filed Feb. 26, 2002; U.S. patent application Ser.No. 10/087,549 entitled “Methods for Decreasing Non-Specific Binding ofBeads in Dual Bead Assays Including Related Optical Biodiscs and DiscDrive Systems” filed Feb. 28, 2002; U.S. patent application Ser. No.10/099,256 entitled “Dual Bead Assays Using Cleavable Spacers and/orLigation to Improve Specificity and Sensitivity Including RelatedMethods and Apparatus” filed Mar. 14, 2002; U.S. patent application Ser.No. 10/099,266 entitled “Use of Restriction Enzymes and Other ChemicalMethods to Decrease Non-Specific Binding in Dual Bead Assays and RelatedBio-Discs, Methods, and System Apparatus for Detecting Medical Targets”also filed Mar. 14, 2002; U.S. patent application Ser. No. 10/121,281entitled “Multi-Parameter Assays Including Analysis Discs and MethodsRelating Thereto” filed Apr. 11, 2002; U.S. patent application Ser. No.10/150,575 entitled “Variable Sampling Control for RenderingPixelization of Analysis Results in a Bio-Disc Assembly and ApparatusRelating Thereto” filed May 16, 2002; U.S. patent application Ser. No.10/150,702 entitled “Surface Assembly For Immobilizing DNA CaptureProbes in Genetic Assays Using Enzymatic Reactions to Generate Signalsin Optical Bio-Discs and Methods Relating Thereto” filed May 16, 2002;U.S. patent application Ser. No. 10/194,418 entitled “Optical DiscSystem and Related Detecting and Decoding Methods for Analysis ofMicroscopic Structures” filed Jul. 12, 2002; U.S. patent applicationSer. No. 10/194,396 entitled “Multi-Purpose Optical Analysis Disc forConducting Assays and Various Reporting Agents for Use Therewith” alsofiled Jul. 12, 2002; U.S. patent application Ser. No. 10/199,973entitled “Transmissive Optical Disc Assemblies for Performing PhysicalMeasurements and Methods Relating Thereto” filed Jul. 19, 2002; U.S.patent application Ser. No. 10/201,591 entitled “Optical Analysis Discand Related Drive Assembly for Performing Interactive Centrifugation”filed Jul. 22, 2002; U.S. patent application Ser. No. 10/205,011entitled “Method and Apparatus for Bonded Fluidic Circuit for OpticalBio-Disc” filed Jul. 24, 2002; U.S. patent application Ser. No.10/205,005 entitled “Magnetic Assisted Detection of Magnetic Beads UsingOptical Disc Drives” also filed Jul. 24, 2002; U.S. patent applicationSer. No. 10/230,959 entitled “Methods for Qualitative and QuantitativeAnalysis of Cells and Related Optical Bio-Disc Systems” filed Aug. 29,2002; U.S. patent application Ser. No. 10/233,322 entitled “CaptureLayer Assemblies for Cellular Assays Including Related Optical AnalysisDiscs and Methods” filed Aug. 30, 2002; U.S. patent application Ser. No.10/236,857 entitled “Nuclear Morphology Based Identification andQuantification of White Blood Cell Types Using Optical Bio-Disc Systems”filed Sep. 6, 2002; U.S. patent application Ser. No. 10/241,512 entitled“Methods for Differential Cell Counts Including Related Apparatus andSoftware for Performing Same” filed Sep. 11, 2002; U.S. patentapplication Ser. No. 10/279,677 entitled “Segmented Area Detector forBiodrive and Methods Relating Thereto” filed Oct. 24, 2002; U.S. patentapplication Ser. No. 10/293,214 entitled “Optical Bio-Discs and FluidicCircuits for Analysis of Cells and Methods Relating Thereto” filed onNov. 13, 2002; U.S. patent application Ser. No. 10/298,263 entitled“Methods and Apparatus for Blood Typing with Optical Bio-Discs” filed onNov. 15, 2002; U.S. patent application Ser. No. 10/307,263 entitled“Magneto-Optical Bio-Discs and Systems Including Related Methods” filedNov. 27, 2002; U.S. patent application Ser. No. 10/341,326 entitled“Method and Apparatus for Visualizing Data” filed Jan. 13, 2003; U.S.patent application Ser. No. 10/345,122 entitled “Methods and Apparatusfor Extracting Data From an Optical Analysis Disc” filed on Jan. 14,2003; U.S. patent application Ser. No. 10/347,155 entitled “OpticalDiscs Including Equi-Radial and/or Spiral Analysis Zones and RelatedDisc Drive Systems and Methods” filed on Jan. 15, 2003; U.S. patentapplication Ser. No. 10/347,119 entitled “Bio-Safe Dispenser and OpticalAnalysis Disc Assembly” filed Jan. 17, 2003; U.S. patent applicationSer. No. 10/348,049 entitled “Multi-Purpose Optical Analysis Disc forConducting Assays and Related Methods for Attaching Capture Agents”filed on Jan. 21, 2003; U.S. patent application Ser. No. 10/348,196entitled “Processes for Manufacturing Optical Analysis Discs with MoldedMicrofluidic Structures and Discs Made According Thereto” filed on Jan.21, 2003; U.S. patent application Ser. No. 10/351,604 entitled “Methodsfor Triggering Through Disc Grooves and Related Optical Analysis Discsand System” filed on Jan. 23, 2003; U.S. patent application Ser. No.10/351,280 entitled “Bio-Safety Features for Optical Analysis Discs andDisc System Including Same” filed on Jan. 23, 2003; U.S. patentapplication Ser. No. 10/351,244 entitled “Manufacturing Processes forMaking Optical Analysis Discs Including Successive Patterning Operationsand Optical Discs Thereby Manufactured” filed on Jan. 24, 2003; U.S.patent application Ser. No. 10/353,777 entitled “Processes forManufacturing Optical Analysis Discs with Molded Microfluidic Structuresand Discs Made According Thereto” filed on Jan. 27, 2003; U.S. patentapplication Ser. No. 10/353,839 entitled “Method and Apparatus forLogical Triggering” filed on Jan. 28, 2003; and U.S. patent applicationSer. No. 10/356,666 entitled “Methods For Synthesis of Bio-ActiveNanoparticles and Nanocapsules For Use in Optical Bio-Disc Assays andDisc Assembly Including Same” filed Jan. 30, 2003. All of theseapplications are herein incorporated by reference in their entireties.They thus provide background and related disclosure as support hereof asif fully repeated herein.

CONCLUDING SUMMARY

All patents, provisional applications, patent applications, technicalspecifications, and other publications mentioned in this specificationare incorporated herein in their entireties by reference.

While this invention has been described in detail with reference tocertain preferred embodiments, it should be appreciated that the presentinvention is not limited to those precise embodiments. Rather, in viewof the present optical bio-system disclosure that describes the currentbest mode for practicing the invention, many modifications andvariations would present themselves to those of skill in the art withoutdeparting from the scope and spirit of this invention. The scope of theinvention is, therefore, indicated by the following claims rather thanby the foregoing description. All changes, modifications, and variationscoming within the meaning and range of equivalency of the claims are tobe considered within their scope.

Furthermore, those skilled in the art will recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are also intended to be encompassed by the following claims.

1. A chromatographic optical bio-disc, comprising: a cap portion havinginlet and vent ports formed therein; a first channel layer having afirst set of flow channels formed therein; a chromatographic layerhaving pass through ports formed therein; a second channel layer havinga second set of flow channels formed therein; and a substantiallycircular substrate having a center and an outer edge.
 2. The opticalbio-disc according to claim 1 further comprising a target zone disposedbetween the center and the outer edge of said substrate in fluidcommunication with said first set of flow channels, said second set offlow channels, and said pass through ports.
 3. The optical bio-discaccording to claim 2 further comprising capture agents located withinsaid target zone.
 4. The optical bio-disc according to claim 3 furthercomprising chromatography membranes.
 5. The optical bio-disc accordingto claim 4 wherein said chromatography membranes are placed on said passthrough ports such that when a sample is introduced through the inletport, the sample moves into the first set of flow channels, through thechromatography membranes and the pass through ports, into the second setof flow channels and into the capture zone.
 6. The optical bio-discaccording to claim 5 wherein said chromatography membranes are ionexchange membranes.
 7. The optical bio-disc according to claim 5 whereinsaid chromatography membranes are membrane adsorbers.
 8. The opticalbio-disc according to claim 5 wherein said chromatography membranes havebinders associated therewith.
 9. The optical bio-disc according to claim8 wherein said binders are directed to glycated hemoglobin.
 10. Theoptical bio-disc according to claim 8 wherein said binders are directedto non-glycated hemoglobin.
 11. The optical bio-disc according to claim1 wherein the substrate includes encoded information associatedtherewith, the encoded information being readable by a disc driveassembly to control rotation of the bio-disc.
 12. The optical bio-discaccording to claim 1 further comprising a reflective layer associatedwith said substrate.
 13. The optical bio-disc according to claim 1further comprising an enzyme, wherein the enzyme, when exposed to anenzyme substrate, produces a signal detectable by an incident beam ofelectromagnetic radiation.
 14. An optical bio-disc, comprising: a capportion having inlet and vent ports formed therein; a channel layerhaving a fluidic circuit formed therein; a substantially circularsubstrate having a center and an outer edge; and a micro-chromatographicmatrix formed in said fluidic circuit.
 15. The optical bio-disc of claim14 further comprising an analysis chamber in fluid communication withsaid fluidic circuit.
 16. The optical bio-disc of claim 15 furthercomprising a filter placed within said fluidic circuit.
 17. The opticalbio-disc of claim 16 further comprising a capture agent associated withthe substrate in the analysis zone.
 18. The optical disc of claim 17wherein said micro-chromatographic matrix is formed from weak cationexchange beads.
 19. The optical disc of claim 17 wherein saidmicro-chromatographic matrix is formed from anionic beads.
 20. Theoptical bio-disc of claim 18 wherein said capture agent is haptoglobin.21. The optical bio-disc of claim 20 wherein said analysis chamber ispre-loaded with a signal agent.
 22. The optical bio-disc of claim 21wherein said signal agent is an antibody.
 23. The optical bio-disc ofclaim 22 wherein said antibody is labeled with a tag.
 24. The opticalbio-disc of claim 23 wherein said tag is detectable by an optical discreader.
 25. The optical bio-disc of claim 23 wherein said tag isselected from the group comprising an enzyme, a fluorescent particle, afluorescent dye, a luminescent dye, and a luminescent particle.
 26. Amethod of using the optical disc according to claim 24 for testing theamount of hemoglobin Alc in a hemoglobin test sample, said method ofusing comprising the steps of: depositing the test sample into the discthrough the inlet port; rotating said disc at a predetermined speed andtime to allow said test sample to move through saidmicro-chromatographic matrix allowing non-glycated hemoglobin present inthe sample to bind to said micro-chromatographic matrix; continuing saidrotating step to move said test sample through said filter, and intosaid analysis chamber; incubating the test sample to allow any glycatedhemoglobin present in the sample to bind with said capture agent andallow said signal agent to bind with said glycated hemoglobin; washingsaid analysis chamber to remove unbound signal agents; and scanning saidanalysis chamber with a beam of electromagnetic radiation to determinethe amount of signal agents bound to the glycated hemoglobin.
 27. Themethod according to claim 26 further comprising the step of calculatingthe amount of glycated hemoglobin present in the sample based on theamount of bound signal agents.
 28. A method of making a chromatographicoptical bio-disc, said method comprising the steps of: providing asubstrate having a center and an outer edge; encoding information on aninformation layer associated with the substrate, the encoded informationbeing readable by a disc drive assembly to control rotation of the disc;forming a target zone in association with the substrate, the target zonedisposed at a predetermined location relative to the center of thesubstrate; depositing a capture agent on the target zone; forming a flowchannel in fluid communication with the target zone; and forming amicro-chromatographic matrix within the flow channel.
 29. A method ofmaking a chromatographic optical bio-disc, said method comprising thesteps of: providing a substrate having a center and an outer edge;encoding information on an information layer associated with thesubstrate, the encoded information being readable by a disc driveassembly to control rotation of the disc; forming a target zone inassociation with the substrate, the target zone disposed at apredetermined location relative to the center of the substrate;depositing a capture agent on the target zone; providing a cap portionhaving an inlet port and a vent port formed therein; providing a firstchannel layer having a first set of flow channels formed therein;providing a chromatographic layer having pass through ports formedtherein; providing a second channel layer having a second set of flowchannels formed therein; forming a chromatography membrane over saidpass through ports; and assembling the optical bio-disc such that saidtarget zone is in fluid communication with said second set of flowchannels, said pass through ports, said first set of flow channels, saidinlet port, and said vent port.
 30. An optical assay disc implemented toperform any of the methods recited in either claim
 26. 31. Use of anoptical analysis disc to perform any of the methods recited in eitherclaim
 26. 32. An optical disc assembly made to perform any of themethods recited in either claim
 26. 33. An optical bio-disc systemadapted to operate the optical assay disc recited in claim
 30. 34. Anoptical bio-disc system adapted to read information stored on theoptical assay disc recited in claim
 30. 35. An optical bio-disc systemadapted to write information relating to results of an assay onto theoptical assay disc recited in claim
 30. 36. An optical bio-disc systemadapted to display on a monitor information relating to results of anassay conducted in association with the optical assay disc recited inclaim
 30. 37. An optical bio-disc system adapted to receive the opticalassay disc recited in claim 30 and facilitate the performance of anassay associated with said optical assay disc.
 38. An optical bio-discsystem adapted to operate the optical analysis disc recited in claim 31.39. An optical bio-disc system adapted to read information stored on theoptical analysis disc recited in claim
 31. 40. An optical bio-discsystem adapted to write information relating to results of an assay ontothe optical analysis disc recited in claim
 31. 41. An optical bio-discsystem adapted to display on a monitor information relating to resultsof an assay conducted in association with the optical analysis discrecited in claim
 31. 42. An optical bio-disc system adapted to receivethe optical analysis disc recited in claim 31 and facilitate theperformance of an assay associated with said optical analysis disc. 43.An optical bio-disc system adapted to operate the optical disc assemblyrecited in claim
 32. 44. An optical bio-disc system adapted to readinformation stored on the optical disc assembly recited in claim
 32. 45.An optical bio-disc system adapted to write information relating toresults of an assay onto the optical disc assembly recited in claim 32.46. An optical bio-disc system adapted to display on a monitorinformation relating to results of an assay conducted in associationwith the optical disc assembly recited in claim
 32. 47. An opticalbio-disc system adapted to receive the optical disc assembly recited inclaim 32 and facilitate the performance of an assay associated with saidoptical disc assembly.